Epicholesterol dehydrogenase

ABSTRACT

The present invention relates to a process for producing a cholesterol-reduced substance obtained by converting cholesterol in a substance to epicholesterol, as well as to a novel cholesterol oxidase and a novel epicholesterol dehydrogenase which are used in the process, a process for production of these enzymes and a method for the production of epicholesterol with the use of the above mentioned epicholesterol dehydrogenase.

This application is a Divisional application of application Ser. No.193,174, filed as PCT/JP93/00771, Jun. 8, 1993, now U.S. Pat. No.5,503,988.

TECHNICAL FIELD

The present invention relates to a process for producing acholesterol-reduced substance; a novel cholesterol oxidase and a novelepicholesterol dehydrogenase to be used in the process; a process forproducing these enzymes; and a process for producing epicholesterol withthe use of the epicholesterol dehydrogenase.

It has been reported that excessive intake of foods having a highcholesterol content causes increase in a concentration of cholesterol inblood serum, and that a high concentration of serum cholesterol is animportant risk factor for heart disease (Dairy Council Digest, 60 (2),7, 1989). Thus, for the purpose of providing low-cholesterol foods andlow-cholesterol feed, methods for the selectively reducing cholesterollevel in foods and feed are in demand. In addition, simple and safeprocess for producing an epicholesterol having a high purity is also indemand. Highly purified epicholesterol is useful in physiologicalresearches.

PRIOR ART

It is known that cholesterol-reduced substances may be obtained byextracting cholesterol from foods with hexane or acetone (JapanesePublished Examined Patent Application No. 42944/71 and JapanesePublished Unexamined Patent Application No. 19062/72) and bysupercritical carbon dioxide extraction (Japanese Published UnexaminedPatent Application Nos. 135847/84, 167035/90 and 98541/91).

It is also known that cholesterol-reduced butter may be obtained byadsorbing cholesterol using polymer-supported digitonin J. Agric. FoodChem., 38 (9), 1839 (1990)!.

It is also known that cholesterol-reduced food may be obtained by addingβ-cyclodextrin to the egg yolk solutions or milk products, and thenseparating insoluble complexes of cholesterol and β-cyclodextrin bycentrifugation (Japanese Published Unexamined Patent Application Nos.252259/89, 98553/91 and 49647/91).

In the above mentioned extraction and adsorption methods, lipids andflavor components are also extracted in addition to cholesterol, andthus the quality of foods is deteriorated.

On the other hand, a method of degrading cholesterol in foods withmicroorganisms Japanese Published Unexamined Patent Application No.267231/88, J. of Food. Science, 53 (2), 659 (1989)! and a method ofconverting cholesterol with cholesterol reductase to coprostanol (U.S.Pat. No. 4,921,710) are known.

Treatment with phospholipase (Japanese Published Unexamined PatentApplication No. 49414/93) and treatment with protease or lipase(Japanese Published Unexamined Patent Application No. 76311/93) areknown to promote the above-mentioned treatment with microorganisms orenzymes.

In addition, a cholesterol oxidase which oxidizes cholesterol to4-cholesten-3-one (EC 1.1.3.6), a cholesterol oxidase produced byBasidiomycetes which oxidizes cholesterol to 5-cholesten-3-one (JapanesePublished Unexamined Patent Application No. 48159/85), a cholesteroldehydrogenase which oxidizes cholesterol to cholestenone (JapanesePublished Examined Patent Application No. 18064/90, Japanese PublishedUnexamined Patent Application No. 56090/78) and a 3α-hydroxysteroiddehydrogenase which oxidizes the 3α-hydroxyl group to cholic acid (EC1.1.1.50) are known.

It is known that epicholesterol is scarcely absorbed through intestinesJournal of Biological Chemistry, 206, 757 (1954)!. It is not known thatcholesterol-reduced substances can be produced by converting cholesterolin foods into epicholesterol. Epicholesterol dehydrogenase whichoxidizes epicholesterol to cholestenone in the presence of NAD(P) is notknown. Furthermore, cholesterol oxidase which is produced bymicroorganisms belonging to the genus Botrytis, and which oxidizescholesterol to 5-cholesten-3-one is not known. On the other hand, achemical method for the conversion of cholesterol to epicholesterol isknown Journal of Organic Chemistry 40 (9), 1361, (1975)!.

DISCLOSURE OF THE INVENTION

The present invention provides a novel process for producing acholesterol-reduced substance. The present invention also provides anovel epicholesterol dehydrogenase and a novel cholesterol oxidase to beused in the process for producing the cholesterol-reduced substance; aprocess for producing the novel enzymes; and a process for producingepicholesterol with the use of the novel epicholesterol dehydrogenase.

According to the present invention, cholesterol-reduced substances maybe prepared without deteriorating the quality of cholesterol-containingsubstances by converting the cholesterol (cholest-5-en-3β-ol) in thesubstances to epicholesterol (cholest-5-en-3α-ol) which is poorlyabsorbed through intestines.

A more detailed description of the process for producingcholesterol-reduced substances of the present invention is given below.

The substances to be used in the present invention comprise a productselected from meat, an egg, milk and seafood; processed and cooked foodcontaining the product; or feed for animals, livestock, fish farming,etc.

As the process to be used to convert cholesterol in the substances toepicholesterol, either of a biochemical process or a chemical processmay be used.

As the biochemical process, mention may be made of a process in whichthe cholesterol is converted to cholestenone, and further toepicholesterol, by the action of enzymes. Specifically, the enzymeshaving the following activity are added to the cholesterol-containingsubstances.

(1) In case of using two enzymes, cholesterol oxidase and epicholesteroldehydrogenase;

Treatments A and B are simultaneously carried out, or alternativelyTreatment A is followed by Treatment B.

Treatment A: treatment with cholesterol oxidase

    Cholesterol+O.sub.2 →Cholestenone+H.sub.2 O.sub.2

Treatment B: treatment with epicholesterol dehydrogenase

    Cholestenone+NAD(P)H→epicholesterol+NAD(P)

In case of using two enzymes, cholesterol dehydrogenase andepicholesterol dehydrogenase:

Treatments C and D are simultaneously carried out, or Treatment C isfollowed by Treatment D.

Treatment C: treatment with cholesterol dehydrogenase

    Cholesterol+NAD(P)→Cholestenone+NAD(P)H

Treatment D: treatment with epicholesterol dehydrogenase

    Cholestenone+AND(P)H→epicholesterol+AND(P)

The enzymes having the above-mentioned activities include, for example,purified enzymes, crude enzymes, microbial cells having those enzymeactivities, and a treated matter of those microbial cells.

The enzymes having the above-mentioned activities (hereinafter referredto as the enzyme source) may be added to the substance as a powder.Preferably, they are dissolved in water and the resulting aqueoussolution is added. Also, if necessary, coenzymes such as NAD(P) andNAD(P)H, or enzymes such as phospholipase, lipase and protease may beadded together with the enzymes.

In case of reducing cholesterol in eggs, the enzyme source is injectedinto the whole egg, or is mixed with the obtained liquid egg yolk orliquid whole egg. In case of reducing cholesterol in beef, pork, muttonand chicken, the enzyme source is mixed with the minced meat, sprayed onthe sliced meat, or injected into the blocked meat. Alternatively, theenzyme source is injected into the animal within 1 hour prior itsslaughtering. In case of reducing cholesterol in milk, the enzyme sourceis mixed with the milk, or alternatively milk is passed through theenzyme source-immobilized carrier. In addition, the enzyme source may beadded during cooking of the egg, meat, milk or seafood.

Likewise, in case of reducing cholesterol in feed for animals, livestockand fish farming, in accordance with the steps for the incorporation offeed ingredients, treatment conditions and amounts of the enzyme addedare arbitrarily selected to carry out the enzymatic conversion toepicholesterol.

In the above mentioned enzymatic treatments, the treatment conditions(temperature, time, pH) and amounts of the enzyme added are arbitrarilyselected to perform the enzymatic conversion to epicholesterol inaccordance with the production steps of the cholesterol-containingsubstance, and the treatment is usually carried out at a reactiontemperature of 5°-70° C. and at pH 4-8, for 30 minutes to 200 hours. Theamount of each of the enzymes, cholesterol oxidase, cholesteroldehydrogenase and epicholesterol dehydrogenase to be used is 1-10⁴units, preferably 10-10³ units, per gram of cholesterol. If necessary,NAD(P) or NAD(P)H is added at an amount of 1×10⁻⁴ -20 g per gram ofcholesterol. Also, if necessary, any of the various phospholipases isadded at an amount of 1×10⁻¹ to 1×10⁵ units per gram of phospholipid.

As the cholesterol oxidase to be used in the present invention, onederived from a microorganism belonging to the genus Botrytis, onederived from the genus Basidiomycetes (Japanese Published ExaminedPatent Application No. 48159/85), and one derived from a microorganismbelonging to the genus Brevibacterium, Nocardia, Pseudomonas orStreptomyces may be used. The latter cholesterol oxidases may becommercially available from Sigma Co.

As the cholesterol dehydrogenase to be used in the present invention,one derived from a microorganism belonging to the genus Nocardia,Alcaligenes or Proteus (Japanese Published Examined Patent ApplicationNo. 18064/90), and one derived from animal liver (Japanese PublishedUnexamined Patent Application No. 56090/78) can be used.

As the epicholesterol dehydrogenase to be used in the present invention,any enzyme can be used so long as it exhibits activity to reducecholestenone to epicholesterol. For example, an enzyme derived from amicroorganism belonging to the genus Mycobacterium is mentioned below.

On the other hand, as the chemical method used to convert cholesterol toepicholesterol, mention may be made of the method of Houminer, et al. J.Org. Chem. 40 (9), 1361 (1975)!. For example, bromine may be added to adried or dried-ground cholesterol-containing substance to convert thecholesterol into cholesterol dibromide, and then cholesterol dibromideis oxidized to dibromocholestane-3-one, and reduced to epicholesterol.By separation of the solids from the reaction solution by filtration,centrifugation, etc. and then drying if necessary, a cholesterol-reducedsubstance may be obtained.

The epicholesterol dehydrogenase of the present invention is a novelenzyme, and its physicochemical properties and method of productionthereof are as follows.

(a) Action:

The enzyme catalyzes the following reaction.

    Epicholesterol+NAD(P)⃡Cholestenone+NAD(P)H

(b) Substrate specificity:

The enzyme acts specifically on epicholesterol; does not act oncholesterol.

(c) Optimum pH:

The optimum pH for the production of cholestenone when epicholesterol isused as the substrate, is 8-12. The optimum pH when epicholesterol isproduced from 5-cholesten-3-one, is 4-5. Determined at 37° C. usingvarious pH buffer, 0.1M acetate/hydrochloride buffer (pH 2-4), 0.1 Mphosphate/citrate buffer (pH 4-7), 0.1 M Tris-HCl buffer (pH 7-9), and0.1 M glycine/sodium hydroxide buffer (pH 9-12), each containing 1 mMdithiothreitol!.

(d) Stable pH:

The enzyme is stable at pH 4-12, determined by mixing the enzymesolution with any of the various pH buffers, allowing the mixture tostand at 37° C. for 60 minutes, and determining the residual activity.

(e) Determination of titer:

With 0.1 ml of a 1 mM epicholesterol micelle solution containing 0.33%Triton X-100 are mixed 0.3 ml of 20 mM Tris-HCl buffer (pH 8.0)containing 1 mM dithiothreitol, 0.1 ml of a 10 mM NAD solution, and 0.01ml of 50 mM magnesium chloride. 0.05 ml of the enzyme solution is addedto the mixture, and the mixture is allowed to stand at 37° C. for 2hours. 0.05 ml of chloroform is added and sterol is extracted to stopthe reaction.

Cholestenone produced in the reaction mixture is determined with highperformance liquid chromatography. A quantitative determination is madeusing an ODS column (Inertsil ODS-2 column, 4.6×250 mm, product of GLScience Co.), using methanol as the mobile phase. As a control, anenzyme which has been thermally inactivated in advance is used, and thecholestenone in the reaction product is determined in the same manner.The enzyme activity which produces 1 μmol of cholestenone per minute isdefined as 1 unit.

(f) Optimum temperature range: 40°-50° C. At pH 8.0, the activityincreases up to 50° C.

(g) Temperature stability: After heating at 50° C., pH 8.0 for 10minutes, more than 80% of the original activity remained.

(h) Influence of inhibitors, metal ions:

When the enzyme activity without adding an inhibitor is defined as 100%,then the residual activities when 1 mM p-chloromercury phenylsulfonate,iodoacetic acid or ethylenediamine tetraacetate is added, and themixture is allowed to stand for 2 hours at pH 8.0, 37° C. are 7%, 67%and 71%, respectively. The activity of the present enzyme is intensifiedin the presence of 0.1-10 mM magnesium ion or manganese ion, comparedwith that in the absence of the ions.

(i) Method of purification:

The culture of organisms producing epicholesterol dehydrogenase iscentrifuged and the harvested cells are suspended in 0.02M Tris-HClbuffer (pH 7.5) containing 1 mM dithiothreitol. The cells are disruptedby ultrasonication, and the solids are removed by centrifugation toobtain a crude enzyme solution. The crude enzyme solution is dialyzedfor 24 hours against 0.02M Tris-HCl buffer (pH 7.5) containing 1 mMdithiothreitol, and applied to a DEAE-Sepharose fast flow (product ofPharmacia Co.) equilibrated with the same buffer. Next, elution isperformed with a linear sodium chloride gradient from 0 to 0.3M in thesame buffer, and the active fractions are collected. Then, the enzymesolution is passed through a gel filtration column (Superose 6, productof Pharmacia Co.) equilibrated with a 0.02 M Tris-HCl buffer containing0.2 M sodium chloride. The elution is performed with the same buffer andthe active fractions are collected. The active fractions arerechromatographed on the same gel filtration column, and the resultingactive fractions are obtained as the purified sample.

(j) Molecular weight

The present enzyme preparation is sonicated for 30 seconds in a buffercontaining 2% Triton X-100, and subjected to gel filtration with highperformance liquid chromatography (Superose 6, product of PharmaciaCo.). The molecular weight of the present enzyme is determined to beapproximately 260,000.

(k) The coenzyme of the present enzyme is β-nicotinamide adeninedinucleotide (NAD)

The present enzyme is regarded as novel due to its properties mentionedabove, and it is possible to produce epicholesterol from acholesterol-containing substrate by the enzymatic activity of thepresent enzyme.

The term "cholesterol-containing substrate" comprises an aqueoussuspension of cholesterol, an aqueous micelle solution of cholesterol,or an aqueous solution in which a cholesterol-containing organic solventlayer is emulsified.

In order to convert cholesterol to epicholesterol, the biochemicalprocesses involving Treatment A and Treatment B, or Treatment C andTreatment D, which are the process for producing a cholesterol-reducedsubstance as mentioned above may be carried out. The enzyme source whichis in the form of either powders or an aqueous solution is added to acholesterol-containing substrate. Alternatively, thecholesterol-containing substrate is passed through an enzymesource-immobilized carrier. If necessary, a coenzyme such as NAD(P) andNAD(P)H may be used in combination therewith.

For the enzyme treatment, the treatment conditions (temperature, time,pH) and the amount of enzyme added are selected so as to allow theenzymatic conversion to epicholesterol, but generally the treatment iseffected at a temperature of 10°-50° C. and at a pH of 4-8, for 30minutes to 48 hours. The amount of each of the enzymes, cholesteroloxidase, cholesterol dehydrogenase and epicholesterol dehydrogenase tobe used is 1-10⁴ units, preferably 10-10³ units per gram of cholesterol.If necessary, NAD(P) or NAD(P)H may be added at 1×10⁻⁴ -20g per gram ofcholesterol.

The enzyme-treated cholesterol-containing substrate may, if necessary,be subjected to filtration, centrifugation, etc. to collect the desiredepicholesterol.

A method for the production of the novel epicholesterol dehydrogenase isdescribed hereinafter.

Any microorganism including variants and mutants can be used for theproduction of the epicholesterol dehydrogenase, so long as it belongs tothe genus Mycobacterium and is capable of producing epicholesteroldehydrogenase. A specific example of a microorganism belonging to thegenus Mycobacterium and being capable of producing epicholesteroldehydrogenase is Mycobacterium sp. EPI-40.

Mycobacterium sp. EPI-40 was newly isolated from soil by the presentinventors, and its mycological properties are as follows.

(a) Morphology

(1) Shape and size of cells: rod Diameter: 0.5-1.2 μm, Length: 1.5-5.0μm

(2) Polymorphism of cells: Polymorphic. Short rods to long rods.Branched form is rarely observed.

(3) Mobility: not observed

(4) Sporulation: not observed

(b) Growth conditions in various media

(1) Nutrient agar plate culture

1) Growth appearance: Colonies have irregular form with undulate marginand rough surface.

2) Color: Ivory

3) Gloss: none

4) Diffusive pigment: not observed

(2) Nutrient broth culture

1) Surface growth: none

2) Turbidity: positive

(3) Bouillon gelatin culture

1) Growth appearance: grown on surface of the medium

2) Liquefaction of gelatin: an entire medium was liquefied.

(4) Action on litmus milk

1) Reaction: alkaline

2) Coagulation: negative

3) Liquefaction: negative

(c) Physiological properties

(1) Gram staining: Cells are negatively stained when cultured for 18hours on yeast extract nutrient agar medium. Cells show: gram-positivetype reaction with 3% potassium hydroxide test.

(2) Reduction of nitrates: positive

(3) Denitrafication reaction: negative

(4) MR test: negative

(5) VP test: negative

(6) Indole production: negative

(7) Hydrogen sulfide production: negative

(8) Starch hydrolysis: positive

(9) Utilization of citric acid: Koser's method: positive Christensen'smethod: positive

(10) Utilization of inorganic nitrogen: nitrates (positive) ammoniumsalts (positive)

(11) Pigment production: negative

(12) Urease: negative

(13) Oxidase: negative

(14) Catalase: positive

(15) Growth range:

1) pH: pH 5.0-pH 11.0

2) Temperature: 12°-38° C.

(16) Attitude towards oxygen: aerobic

(17) Production of acid or gas from carbohydrates:

    ______________________________________               Acid production                         Gas production    ______________________________________    L-Arabinose  -           -    D-Xylose     -           -    D-Glucose    + (weak)    -    D-Mannose    + (weak)    -    D-Fructose   + (weak)    -    D-Galactose  -           -    Maltose      -           -    Sucrose      -           -    Lactose      -           -    Trehalose    -           -    D-sorbit     -           -    D-mannit     -           -    Inosit       -           -    Glycerine    -           -    Starch       -           -    ______________________________________

(d) Chemotaxonomic properties

(1) Amino acid composition of cell wall peptidoglycan: Meso-form ofdiaminopimelic acid as the diamino acid of the cell wall peptidoglycan.

(2) Cellular lipids

1) Isoprenoid quinones: menaquinone which has 9 isopren units with onesaturation (MK-9 (H₂)) is predominant.

2) Fatty acids (including mycolic acid): mycolic acids (complex type)are present.

According to Bergey's Manual of Systematic Bacteriology (Vol. 2, 1986,Section 16: The Mycobacteria), the strain is identified to the genusMycobacterium, on the grounds that it is Gram-positive (3% potassiumhydroxide test); that it takes a polymorphic shape of long rods or shortrods; that it grows aerobically and does not grow anaerobically; that ithas the meso-form of diaminopimelic acid as the diamino acid compositionof its cell wall; that it has the complex type of mycolic acid in thecellular lipids; and that it contains MK-9 (H₂) as its major quinone.The strain was named Mycobacterium sp. EPI-40, and has been depositedwith National Institute of Bioscience and Human-Fermentation ResearchTechnology, Agency of Industrial Science and Technology (FRI) in Japanas FERM BP-4306.

As the medium to be used for the culturing of the epicholesteroldehydrogenase-producing microorganism of the present invention, anysynthetic or natural medium can be used so long as it contains a carbonsource, a nitrogen source, an inorganic substance, etc. As the carbonsource to be used, various carbohydrates such as glucose, glycerol,molasses and epicholesterol can be used and it is preferably used at anamount of about 5-70 g/l. As the nitrogen source to be used, ammoniumsulfate, ammonium phosphate, ammonium carbonate and ammonium acetate, aswell as a nitrogen-containing organic substance such as peptone, yeastextract, corn steep liquor, caseine hydrolysate and beef extract can beused and it is preferably used at an amount of about 5-20 g/l. As theinorganic substance to be used, sodium chloride, potassium dihydrogenphosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesiumchloride and the like can be used and it is preferably used at an amountof about 0.05-5 g/l. In addition, a surfactant may be added ifnecessary. The culturing is carried out under aerobic conditions byshaking culture or aeration-agitation submerged culture. The culturingtemperature may be a temperature at which the cells can grow and produceepicholesterol dehydrogenase, and is preferably 25°-37° C. The culturingperiod depends on the culturing conditions, but may be a period whichallows the maximum production of epicholesterol dehydrogenase, and isnormally about 3-7 days.

The cholesterol oxidase according to the present invention is a novelenzyme, and its physicochemical properties and method of production areas follows.

(a) Action:

The enzyme catalyzes the following reaction.

    Cholesterol+O.sub.2 →5-choleten-3-one+H.sub.2 O.sub.2

(b) Substrate specificity:

It acts specifically on cholesterol which has a hydroxyl group at the3βposition; does not act on epicholesterol.

(c) Optimum pH: 3-7

(d) Stable pH:

The enzyme is stable at 2-8, determined by allowing the enzyme solutionin any of the various pH buffers to stand at 37° C. for 60 minutes, anddetermining the residual activity.

(e) Determination of titer:

To 0.1 ml of 1 mM cholesterol micelle solution containing 0.33% TritonX-100 is added 0.3 ml of 20 mM phosphate/citrate buffer (pH 6.0), 0.05ml of the enzyme solution is added to the mixture, and the reaction iscarried out at 37° C. for 10 minutes. 0.05 ml of chloroform is addedthereto to extract sterol, stopping the reaction.

Cholestenone in the reaction mixture is determined with a TLC/FIDIATROSCAN (product to Diayatron Co.). As a control, an enzyme which hasbeen thermally inactivated in advance is used, and the reaction productis treated in the same manner. The enzyme activity which produces 1 μmolof cholestenone per minute is defined as 1 unit.

(f) Optimum temperature range: 30 °-60° C. At pH 6.0, the activityincreases up to 60° C.

(g) Temperature stability: After heating at 60° C. for 10 minutes, morethan 80% of the original activity remained.

(h) Influence of inhibitors, metal ions:

When the enzyme activity without adding an inhibitor is defined as 100%,then the residual activities upon addition of 1 mM p-chloromercuryphenylsulfonate, iodoacetic acid or ethylenediamine tetraacetate and thereaction for 10 minutes at pH 6.0, 37° C. are 100%, 89%, and 93%,respectively. The activity of the present enzyme is accelerated in thepresence of 0.1-10 mM iron ion, copper ion or magnesium ion.

(i) Method of purification:

The culture is centrifuged to obtain a supernatant. Ethanol is added tothe supernatant to a concentration of 50 v/v%, and the resultingprecipitate is collected by centrifugation, and suspended in 0.02MTris-HCl buffer (pH 8.0). The suspension is dialyzed against the samebuffer for 24 hours, and the dialyzate is applied to a DEAE-Sepharosefast flow (product of Pharmacia Co.) equilibrated with the same buffer.Then, elution is performed with a linear sodium chloride gradient from0-1.0 M in the same buffer and the active fractions are collected. Then,the enzyme solution is passed through a gel filtration column (Superose6, product of Pharmacia Co.) equilibrated with 0.02M Tris-HCl buffercontaining 0.2 M sodium chloride. The elution is carried out with thesame buffer and the active fractions are collected. The active fractionsare rechromatographed on the same gel filtration column, and theresulting active fractions are used as the purified sample.

(j) Molecular weight

The molecular weight of the present enzyme is determined to beapproximately 45,000 by gel filtration with high performance liquidchromatography (Superose 6, product of Pharmacia Co.).

The present enzyme is a novel cholesterol oxidase due to its propertiesmentioned above, and it is possible to convert cholesterol into5-cholesten-3-one, over a wide range of temperature and pH by utilizingthe present enzyme.

A process for producing the present enzyme is given below.

Any microorganism including variants and mutants can be used for theproduction of the present enzyme, so long as it belongs to the genusBotrytis and is capable of producing the cholesterol oxidase. A specificexample of a microorganism which belongs to the genus Botrytis and iscapable of producing cholesterol oxidase is Botrytis cinerea CO-33.

The mycological properties of Botrytis cinerea CO-33 are as follows.

On a malt extract agar medium, the colony appears gray to dark greenishgray at 25° C. The hyphae are septate, smooth and colorless to lightbrown, and are well branched. A conidiophore extends upright from thehyphae, reaching a length of 2 mm or longer. Its width is 15-30 μm, andit is smooth and septate. The conidiophore is solitary, with its upperregion sparsely branched, and many conidia forming on each tip thereof.The conidial ontogeny is holoblastic and a type of a botryoseblastospore. The conidia are colorless to light yellowish brown, 6-26 μmin length, 4-11 μm in width and smooth, and are elliptical to obovate,pear-shaped in almost all of them and sometimes of indefinite shape. Thepresent strain is observed only as the above described anamorph, and notas a teleomorph. As a result of the above mentioned observations, thepresent strain was identified as Botrytis cinerea. The mycologicalproperties of Botrytis cinerea are described in detail by M. B. Ellis in"Dematiaceous Hyphomycetes", 1971, on page 179. The present strain wasnamed "Botrytis cinerea CO-33", and has been deposited with the FRI, asFERM BP-4307.

As the medium to be used for the culturing of the novel cholesteroloxidase-producing microorganism of the present invention, any syntheticor natural medium can be used, so long as it contains a carbon source, anitrogen source, an inorganic substance, etc. As the carbon source to beused, various carbohydrates such as glucose, glycerol, molasses,vegetable juice and starch can be used and it is preferably used at anamount of about 5-70 g/l. As the nitrogen source to be used, ammoniumsulfate, ammonium phosphate, ammonium carbonate, ammonium acetate, aswell as a nitrogen-containing organic substance such as peptone, yeastextract, corn steep liquor, caseine hydrolysate and beef extract can beused, and it is preferably used at an amount of about 5-20 g/l. As theinorganic substance to be used, sodium chloride, potassiumdihydrogenphosphate, dipotassium hydrogen phosphate, magnesium sulfate,magnesium chloride, calcium carbonate and the like can be used and it ispreferably used at an amount of about 0.05-5 g/l. The culturing iscarried out under aerobic conditions by shaking culture oraeration-agitation submerged culture. The culturing temperature may be atemperature at which the cells can grow and produce the novelcholesterol oxidase, and is preferably 20°-30° C. The culturing timedepends on the culturing conditions, but may be a period which allowsthe maximum production of the novel cholesterol oxidase, and is normallyabout 5-8 days.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of gas chromatography analysis of the lipidextracted from a powdered sample of cholesterol-reduced egg yolk,according to Example 9.

BEST MODE FOR CARRYING OUT THE PRESENT INVENTION EXAMPLE 1

Production of epicholesterol dehydrogenase:

Two hundred seventy grams of bouillon granules (product of KyokutoSeiyaku) and 70g of yeast extract (product of Difco Co.) were dissolvedin 10 l of deionized water, the pH was adjusted to 7.0, and the solutionwas poured in 300 ml portions into 2 l-Erlenmeyer flasks. The culturemedium was sterilized at a temperature of 120° C. for 15 minutes, andMycobacterium sp. EPI-40 was inoculated into the medium and cultured byshaking at a temperature of 28° C. for 72 hours.

After completion of the culturing, the cells were collected bycentrifuging 10 l of the culture, washed once with a 0.02M Tris-HClbuffer solution (pH 7.5) containing 1 mm dithiothreitol, and suspendedin the same buffer to make a liquid volume of 200 ml. The cellsuspension was subjected to ultrasonication at 20 KHz for 10 minutes,and the solids were removed by centrifugation to obtain a crude enzymesolution. The crude enzyme solution was dialyzed for 24 hours against0.02M Tris-HCl buffer (pH 7.5), containing 1 mM dithiothreitol, andapplied to a DEAE-Sepharose fast flow (product of Pharmacia Co.)equilibrated with the same type of buffer. Next, the sodium chlorideconcentration was raised in a continuous manner from 0 to 0.3 M forelution.

The solution was passed through a gel filtration column (Superose 6,product of Pharmacia Co.) equilibrated with 0.02 M Tris-HCl buffercontaining 0.2M sodium chloride and 1 mM dithiothreitol, and elution wasperformed with the same buffer with high performance liquidchromatography, to collect the active fractions. The active fractionswere again subjected to the same gel filtration column, and the activefractions were collected by high performance liquid chromatography toobtain the purified enzyme solution. The protein concentration of thepresent enzyme was determined by a protein assay kit (product of BioradCo.), and upon determination of the activity, the specific activity was1.08 units per milligram of protein.

EXAMPLE 2

Production of cholesterol oxidase:

Deionized water was added to a mixture of 2 l of V8 vegetable juice(product of Campbell Co.) and 30g of calcium carbonate until the totalvolume was 10, and the pH was adjusted to 7.2. The mixture was poured,in 300 ml portions, into 2 l-Erlenmeyer flasks. The medium wassterilized at a temperature of 120° C. for 15 minutes, and Botrytiscinerea CO-33 was inoculated and cultured by shaking at a temperature of25° C. for 5 days. After completion of the culturing, 10 l of theculture was centrifuged to obtain a supernatant. Ethanol was added tothe supernatant to 50 v/v%, and the precipitated protein was collectedby centrifugation. The precipitate was suspended in 0.02 M Tris-HClbuffer (pH 7.5) containing 1 mM dithiothreitol and dialyzed against thesame buffer for 24 hours. The active fractions were adsorbed onto aDEAE-Sepharose fast flow (product of Pharmacia Co.) equilibrated withthe same buffer. Then, elution is performed with a linear sodiumchloride gradient from 0 to 1.0 M.

The enzyme solution was passed through a gel filtration column (Superose6, product of Pharmacia Co.) equilibrated with 0.02 M Tris-HCl buffercontaining 0.2M sodium chloride and 1 mM dithiothreitol, and elution wasperformed with the same buffer with high performance liquidchromatography to collect the active fractions. The active fractionswere rechromatographed on the same gel filtration column with highperformance liquid chromatography to obtain the purified enzymesolution. The protein concentration of the present enzyme was determinedwith a protein assay kit (product of Biorad Co.), the specific activitywas determined to be 10.4 units per milligram of protein.

EXAMPLE 3

To 10 ml of 1 mM cholesterol micelle solution containing 0.33% TritonX-100 were added 30 ml of 100 mM phosphate/citrate buffer (pH 4.0)containing 1 mM dithiothreitol and 10 ml of 10 mM NADH solution, and tothe mixture was added 1.0 unit of the cholesterol oxidase obtained inExample 2 and 0.2 unit of the epicholesterol dehydrogenase obtained inExample 1, and the reaction was conducted at 37° C. for 3 hours, 10 mlof chloroform was added thereto to extract sterol. The resultingepicholesterol was determined by high performance liquid chromatography.A quantitative determination was made using an ODS column (InertsilODS-2 column, 4.6×250 mm, product of GL Science Co.) with methanol asthe mobile phase, and the conversion rate to epicholesterol was found tobe 92.5%.

EXAMPLE 4

To 10 ml of a 1 mM cholesterol micelle solution containing 0.33% TritonX-100 were added 30 ml of 100 mM Tris-HCl buffer (pH 8.0) containing 1mM dithiothreitol and 10 ml of 30 mM NAD solution, and to the mixturewas added 200 units of cholesterol dehydrogenase (CHDH "Amano" II,product of Amano Seiyaku). The reaction was conducted at 37° C. for 2hours. The cholesterol dehydrogenase activity was determined accordingto the method described in Japanese Published Examined PatentApplication No. 18064/90. After completion of the reaction, 10 ml ofhexane was added to the mixture to extract sterol, and the sterolsolution was dried. Also, the obtained sterol was dispersed in 30 ml of100 mM phosphate/citrate buffer (pH 4.0) containing 0.33% Triton X-100and 1 mM dithiothreitol, and then 10 ml of a 10 mM NADH solution wasmixed therewith, 0.2 unit of the epicholesterol dehydrogenase obtainedin Example 1 was added thereto, and the reaction was conducted at 37° C.for 2 hours. After completion of the reaction, 10 ml of chloroform wasadded thereto, and the sterol was extracted. Following the method inExample 3, the conversion rate of the obtained sample to epicholesterolwas found to be 58.8%.

EXAMPLE 5

To 5 grams of commercial hen's egg yolk was added 5 ml of water, the pHwas adjusted to 4, and to the mixture were added 20 units of thecholesterol oxidase obtained in Example 2, 5 units of the epicholesteroldehydrogenase obtained in Example 1, and 0.7 g of NADH. The reaction wasconducted at 37° C. for 5 hours. After the reaction, the pH was adjustedto 7. The treated egg yolk was lyophilized, and the lipid fraction ofthe obtained sample was extracted with a solvent(chloroform:methanol=2:1). Following the method in Example 3, theconversion rate of the obtained sample to epicholesterol was found to be72.3%. Thus, egg yolk with a 72.3% reduction of cholesterol wasobtained.

EXAMPLE 6

Five grams of commercial minced beef was adjusted to pH 4, then 25 unitsof phospholipase D, 30 units of the cholesterol oxidase obtained inExample 2, 0.2 unit of the epicholesterol dehydrogenase obtained inExample 1 and 17 mg of NADH were added thereto, and the reaction wasconducted at 37° C. for 5 hours. After completion of the reaction, thepH was adjusted to 7. The treated beef was lyophilized, and the lipidfraction of the obtained sample was extracted with a solvent(chloroform:methanol=2:1). Following the method in Example 3, theconversion rate to epicholesterol in the beef was found to be 23.1%.Thus beef with a 23.1% reduction of cholesterol was obtained.

EXAMPLE 7

To 50 ml of commercial milk were added 100 units of the cholesteroloxidase obtained in Example 2, 1.0 unit of the epicholesteroldehydrogenase obtained in Example 1 and 0.1 g of NADH, and the reactionwas conducted at pH 4, 37° C. for 5 hours. After completion of thereaction, the pH was adjusted to 7. The treated milk was lyophilized,and the lipid fraction of the obtained sample was extracted with asolvent (chloroform:methanol=2:1) for collection. Following the methodin Example 3, the conversion rate to epicholesterol in the milk wasfound to be 15.3%.

Thus milk with a 15.3% reduction of cholesterol was obtained.

EXAMPLE 8

To 5 grams of commercial hen's egg yolk was added 5 ml of water, and thepH was adjusted to 8. 200 units of cholesterol dehydrogenase (CHDH"Amano" II, product of Amano Seiyaku) and 0.3g of NAD were addedthereto, and the reaction was conducted at 37° C. for 3 hours. After thecompletion of the reaction, the pH was adjusted to 4, and 9.0 units ofthe epicholesterol dehydrogenase obtained in Example 1 and 0.7 g of NADHwere added to the solution, and the mixture was allowed to stand at 37°C. for 4 hours, and the pH was adjusted to 7.

The treated egg yolk was lyophilized, and the lipid fraction of theobtained sample was extracted with a solvent (chloroform:methanol=2:1).Following the method in Example 3, the conversion rate to epicholesterolin the egg yolk was found to be 10.5%. Thus, egg yolk with a 10.5%reduction of cholesterol was obtained.

EXAMPLE 9

Hen's egg yolk was filtered with gauze and then lyophilized to obtain apowdered sample. 20g of the sample was dispersed in 140 ml of ether withheating at 40° C., 660 mg of anhydrous sodium acetate was dissolvedtherein, and 80 ml of acetic acid containing 3.5 ml of bromine (Br₂) wasdropwise added thereto. The solution was then immediately cooled on iceand dispersed in 1 l of water, and then was centrifuged to obtain aprecipitate.

The precipitate was dispersed in 260 ml of acetic acid, and to theresulting dispersion was added 4.2g of sodium dichromate dihydrate (Na₂Cr₂ O₂.2H₂ O). 104 ml of acetic acid which had been heated to 90° C. wasadded thereto, and the mixture was stirred for 5 minutes. It was thencooled on ice for 10 minutes and dispersed in 2 l of water, and thencentrifuged to obtain a precipitate.

The resulting precipitate was dispersed in 110 ml of ethanol, 1.2 g ofsodium borohydride (NaBH₄) was added thereto, and the mixture wasstirred for 3 hours at 25° C. Fifty milliliters of acetic acid was addedthereto to decompose the excess NaBH₄, and 500 ml of water was furtheradded thereto for dispersion. The mixture was centrifuged to obtain aprecipitate. The precipitate was then lyophilized to obtaincholesterol-reduced egg yolk powder.

The conversion rate of cholesterol to epicholesterol was estimated bygas chromatography. The lipid was extracted from the powdered samplewith a chloroform-methanol (2:1) solution. The analysis was effectedwith a TC-1701 gas chromatography column (15 m×0.53 mm, product of GLScience Co.) with a temperature increase of 2° C. per minute from 240°C. to 280° C., and detection was made using a hydrogen flame ionizationdetector. The results are shown in FIG. 1.

In this manner, 48.7% cholesterol-reduced egg yolk was obtained.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to considerablyreduce the amount of cholesterol in foods without impairing their tasteor flavor.

What is claimed is:
 1. Substantially pure epicholesterol dehydrogenasehaving the following physicochemical properties:(a) Action: The enzymecatalyzes the following reaction;

    Epicholesterol+NAD(P)⃡Cholestenone+NAD(P) H

(b) Substrate specificity: the enzyme acts specifically onepicholesterol; does not act on cholesterol; (c) Optimum pH: pH 8-12 forthe oxidation reaction from epicholesterol; pH 4-5 for the reductionreaction from 5-cholesten-3-one; (d) Stable pH; 4-12; (e) Optimumtemperature range: 40°-50° C.; (f) Temperature stability: after heatingat 50° C., pH 8.0 for 10 minutes, more than 80% of the original activityremained; (g) Influence of inhibitors, metal ions: the enzyme activityis inhibited by p-chloromercury phenylsulfonate, iodoacetic acid andethylenediamine tetraacetate; the enzyme activity is accelerated bymagnesium ion and manganese ion; (h) Molecular weight: approximately260,000 (by gel filtration); (i) Coenzyme: β-nicotinamide adeninedinucleotide (NAD).
 2. The substantially pure epicholesteroldehydrogenase according to claim 1, which is produced by a microorganismbelonging to the genus Mycobacterium, wherein said microorganism isdeposited with the Fermentation Research Technology, Agency ofIndustrial Science and Technology in Japan as FERM BP-4306. 3.Substantially pure epicholesterol dehydrogenase produced by a processcomprising the steps of:(a) culturing in a suitable medium amicroorganism belonging to the genus Mycobacterium, wherein saidmicroorganism is capable of producing epicholesterol dehydrogenase inculture, and is deposited with the Fermentation Research Technology,Agency of Industrial Science and Technology in Japan as FERM BP-4306,and (b) recovering said epicholesterol dehydrogenase therefrom.